FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex

The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.     

Inhibition of palmitate‐induced GADD34 expression augments apoptosis in mouse insulinoma cells (MIN6)

Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta‐cells, an event linked to apoptotic loss of β‐cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage‐inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin‐producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA‐activated protein kinase‐like ER‐localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate‐induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2α phosphorylation sensitizes insulin‐producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels. Copyright © 2014 John Wiley & Sons, Ltd.     

Wnts as kidney tubule inducing factors.

Since the discovery that inductive tissue interactions regulate nephrogenesis, one of the aims has been to identify the molecules that mediate this induction. The small size of embryonic tissue has limited the possibilities to identify the inducers biochemically, even though such efforts were directed to study, e.g. neural induction (for a comprehensive review, Saxén and Toivonen, Primary embryonic induction, Academic Press, London, 1962). The rapid progress in molecular biology made it possible to identify genes from minute amounts of tissue and provided techniques to generate recombinant proteins to assay their action in classic experimental systems. This led to the identification of some signals that are involved in primary and secondary inductive interactions during embryogenesis. Here, we will review evidence suggesting that secreted signaling molecules from the Wnt gene family mediate kidney tubule induction.     

5-Hydroxytryptamine stimulates Rb efflux from pancreatic β-cells

The effects of 5-hydroxytryptamine and 5-hydroxytryptophan on ⁸⁶Rb⁺ efflux from prelabelled ob/ob-mouse islets were studied to better understand the cellular mechanisms underlying the effects of 5-hydroxytryptamine and 5-hydroxytryptophan on insulin release. 5-Hydroxytryptophan (4 mM) had no effect on ⁸⁶Rb⁺ efflux either at a low (3 mM) or at a high (20 mM) d-glucose concentration, whereas 5-hydroxytryptamine (4 mM) stimulated ⁸⁶Rb⁺ efflux at both glucose concentrations. These results indicate that 5-hydroxytryptamine may reduce glucose-induced insulin release by inhibiting early steps in the β-cell stimulus-secretion coupling.     

Hyperosmosis enhances radiation and hydroxyurea resistance of Schizosaccharomyces pombe checkpoint mutants through the spindle checkpoint and delayed cytokinesis

The DNA damage and stress response pathways interact to regulate cellular responses to genotoxins and environmental stresses. How these pathways interact in Schizosaccharomyces pombe is not well understood. We demonstrate that osmotic stress suppresses the DNA damage sensitivity of checkpoint mutants, and that this occurs through three distinct cell cycle delays. A delay in G2/M is dependent on Srk1. Progression through mitosis is halted by the Mad2‐dependent spindle checkpoint. Finally, cytokinesis is impaired by modulating Cdc25 expression. These three delays, imposed by osmotic stress, together compensate for the loss of checkpoint signalling.     

Quantitative Determination of Bovine Plasma Fibrinogen by Rocket Immunoelectrophoresis and by Refractometry after Heat-Precipitation

A standard syneresis method for measuring plasma fibrinogen concentrations in cattle was compared with a rocket Immunoelectrophoresis method and with a simple refractometer method; the correlations between the results obtained by the standard method and by the other 2 methods were high (r = 0.94 and 0.89 respectively, p < 0.001). The immunoelectrophoretic method is efficient and simple to perform. In contrast with the standard syneresis method, the other methods allow for the fibrinogen degradation products produced by fibrinolysis in vitro; they can therefore tolerate longer sample storage before analysis. The refractometer method is exceptionally simple and rapid and can be recommended for use under field conditions.     

Seasonal variation in food allergy to apple

The aim of the study was to investigate the possibility of a seasonal variation in reactivity to apples in 27 birch pollen allergic patients. Before and during the birch pollen season 1998, the patients were subjected to double-blind, placebo-controlled food challenges (DBPCFCs) with grated fresh Golden Delicious apple followed by an open food challenge with whole fresh apple. The clinical reactions elicited during the challenges were evaluated both by the patients and the investigators. Moreover, the skin reactivity and the in vitro reactivity to apple were evaluated by skin prick test (SPT), leukocyte histamine release (HR), measurement of specific IgE, and immunoblotting experiments. The sensitivity of the DBPCFC, when compared with the result of the open challenge, was 0.74 (14/19) before the season and 0.80 (16/20) during the season. None of the patients reacted to the blinded challenge without a subsequent reaction to the open challenge. One placebo reaction was registered both before and in season, but not in the same patient. The patient scores of the first positive challenges, and the maximal scores of each combined blinded and open challenge session, were significantly increased during the pollen season (P<0.05). The scores of the open challenge were significantly higher than the scores of the DBPCFC both before the season and during the in-season challenges (P<0.05). Specific IgE against Golden Delicious increased during season (P<0.05), while neither SPT, HR, nor immunoblotting experiments could confirm an increase in reactivity. In conclusion, the results of the oral challenge tests indicated an increase in clinical reactivity to apples during the birch pollen season in birch pollen allergic individuals.